I. Biotin Biosynthesis
Biotin (vitamin H) is an essential nutrient for all living organisms (Eisenberg, M. A., Adv. Enzymol. 38: 317-372 (1973)). It is a basic component of cell metabolism that acts as a cofactor that binds covalently to carboxylases to facilitate the transfer of carboxyl groups during enzymatic carboxylation, decarboxylation and transcarboxylation reactions (Knowles, J. R., Ann. Rev. BioChem. 58: 195-221 (1989)). The chemical structure of the naturally occurring d-isomer of biotin is as follows: ##STR1##
Biotin biosynthesis has been extensively studied in microorganisms, primarily through the isolation and characterization of biotin auxotrophic mutants (Eisenberg, supra). Through this work, four enzymatic steps common to E. coli and other microorganisms for the biosynthesis of biotin from the precursor pimeloyl-CoA have been elucidated (Eisenberg, supra; Pai, C. H., Canad. J. Microbiol. 15: 21-26 (1969); del Campillo-Campbell et al., J. Bacteriol. 94: 2065-2066 (1967)). Analysis of two classes of E. coli mutants, those defective in either the bioC (SEQ ID NO:11) or the bioH gene, suggests that the products of these genes play a role in biotin synthesis, but at steps prior to pimeloyl-CoA. The final common steps of the biotin biosynthetic pathway are as follows: ##STR2##
The first step in this common biotin biosynthetic pathway is the synthesis of 7-keto-8aminopelargonic Acid (KAP) from pimeloyl-CoA and L-alanine. This step is catalyzed by an enzyme known as KAP synthetase, which is encoded by the bioF gene in E. coli (Eisenberg, supra). This gene is part of the E. coli biotin operon, which has been cloned and sequenced (Otsuka, A. J. et al., J. Biol. Chem. 263: 19577-19585 (1988); Genbank accession no. J04423).
The second step in this common biotin biosynthetic pathway is the conversion of KAP into 7,8-Diaminopelargonic Acid (DAP). This step is catalyzed by an enzyme known as DAP aminotransferase, which is encoded by the bioA gene (Eisenberg and Stoner, in Methods in Enzomology 62: 342-347, ed. by McCormick and Wright, pub. by Acad. Press, NY (1979); Stoner and Eisenberg, J. Biol. Chem. 250: 4037-4043 (1975); Stoner and Eisenberg, J. Biol. Chem. 250: 4029-4036 (1975); Eisenberg, supra; Eisenberg and Stoner, J. Bacteriol. 108: 1135-1140 (1971); Pai, C. H., J. Bacteriol. 105: 793-800 (1971)). The bioA gene is also part of the E. coli biotin operon, which has been cloned and sequenced (Otsuka, A. J. et al., supra.; Genbank accession no. J04423).
The third step in this common biotin biosynthetic pathway is the conversion of DAP into desthiobiotin. This step is catalyzed by an enzyme known as desthiobiotin synthetase, which is encoded by the bioD gene (Eisenberg, M. A., Ann. N.Y. Acad. Sci. 447: 335-349 (1985); Cheeseman and Pai, J. Bacteriol. 104: 726-733 (1970); Eisenberg and Krell, J. Biol. Chem. 244: 5503-5509 (1969); Pai, C. H., J. Bacteriol. 99: 696-701 (1969)). The bioD gene is also part of the E. coli biotin operon, which has been cloned and sequenced (Otsuka, A. J. et al., supra.; Genbank accession no. J04423).
The final step in this common biotin biosynthetic pathway involves the addition of sulfur to desthiobiotin and subsequent ring closure to form biotin. These steps are catalyzed by an enzyme known as biotin synthase, which is encoded by the bioB gene (Eisenberg, M. A., Ann. N.Y. Acad. Sci. 447: 335-349 (1985); Pai, C. H., J. Bacteriol. 112: 1280-1287 (1972)).
The biotin biosynthetic pathway in plant cells has also been elucidated (Baldet, P. et al., Eur. J. BioChem 217: 479-485 (1993)). This pathway is very similar to the pathway common to all microorganisms, which is described above, with two additional steps. First, the pathway in plants includes the conversion of pimelic acid to pimeloyl-CoA. This step is catalyzed by an enzyme known as pimeloyl-CoA synthetase. This step may also occur in a number of microorganisms, although it may not be common to all (Gloeckler, R. et al., Gene 87: 63-70 (1990); Eisenberg, M., in "Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology", pp. 544-550, ed. by Neidhardt, F. C. et al., pub. by Amer. Soc. Microbiol., NY (1987); Izumi, Y. et al., in Methods in Enzomology 62: 327-330, ed. by McCormick and Wright, pub. by Acad. Press, NY (1979); Izumi, Y. et al., BioChem. Biopys. Acta 264: 210-213 (1972)).
Secondly, the conversion of desthiobiotin to biotin involves the creation of an intermediate compound, 9-mercaptodethiobiotin (Baldet et al., supra). This intermediate may also occur in microorganisms, as conversion of desthiobiotin into biotin in these organisms is not completely understood and as this compound will support the growth of E. coli bioB mutants (Baldet et al, supra).
II. Biotin as a Nutrient
For higher eukaryotic organisms other than plants and some fungi, biotin is an essential vitamin that must be part of the diet. Biotin deficiencies in animals can have a number of adverse effects, including a reduction in growth rate, alopecia (hair loss), scaly dermatitis, and edema and erythema of the feet (Nutritional Reviews 48: 352-355 (1990); Kopinski, J. S. et al., J. Nutrition 62: 751-759 (1989); Poultry Science 67: 590-595 (1988); Marshall, M. W., Nutrition Today 22-23: 26-29 (1987)). In humans, biotin deficiency has also been associated with a number of genetic and acquired diseases (Marshall, M. W., supra).
In general, plant-based feeds do not contain enough biotin to serve as a sufficient dietary source of this vitamin. This is especially true for stockyard animals such as pigs and chickens (Frigg, M., Poultry Science 63: 750-753 (1983). Enhanced performance has been observed in a number of production animals following biotin supplementation of the normal diet (Kopinski, J. S. et al. British Journal of Nutrition 62:751-789)). As a result, additional biotin is incorporated as a feed supplement into the diet of many animals (Robel, E. J., poultry Science 70: 1716-1722 (1991)).
If biotin production in plants could be increased, the need for additional biotin in animal and human diets from sources other than plants could be reduced or eliminated. However, until the present invention, not enough was known about this pathway in plants, or its regulation, to achieve the objective of increasing biotin production in plants.
One approach for enhancing biotin production that might be considered would be to alter the levels of intermediates or enzymes in the biotin biosynthetic pathway. However, in light of what was previously known, this approach would not have been expected to work because metabolic pathways are typically tightly regulated so that metabolite synthesis remains stable despite fluctuations that may occur in the levels of available pathway intermediates and enzymes. Regulation of metabolite synthesis may involve a variety of mechanisms. Classic examples of mechanisms used to regulate metabolite synthesis in microorganisms include catabolite repression and enzyme induction (Dickson et al. Science 187:27-35 (1975)), feedback inhibition (Stryer, L., "BioChemistry", 2nd ed., pub. by W. H. Freeman and Co., San Francisco, pp. 500-503 (1981)), attenuation (Wu, A. and Platt, T. Proc. Nat. Acad. Sci. U.S. 75:5442 (1978)), and general control (M. Wolfner et al. J. Mol. Biol. 96:273-290)). Some or all of these mechanisms may also be involved in metabolic pathway regulation in plants. Because these pathways are typically tightly regulated through a variety of mechanisms, the effect that increasing the amount of any one enzyme in a pathway would have, if any, upon the final level of the end product (metabolite) synthesized could not ordinarily be predicted.